PROJECT SUMMARY Anxiety disorders are the most common psychiatric illness afflicting 273 million people worldwide. The symptoms of anxiety disorders are highly complex and the causes are poorly understood. Additionally, a substantial number of patients suffering from anxiety disorders also present with depressive-like symptoms, which make the diagnosis and treatment even more complicated. In this project, we propose to utilize the repeated social defeat stress (RSDS) model that induces anxiety or mixed anxiety/depression phenotypes in separate subgroups of mice to investigate neural mechanisms regulating these behaviors. Following RSDS, mice are separated into two subgroups based on their social interaction test behavior: profound avoidance-displaying mice (susceptible) and non-avoidance-displaying mice (resilient). While susceptible mice display several depression-related behaviors, including social avoidance, anhedonia and despair, these depressive abnormalities are absent in resilient mice. However, RSDS induces severe anxiety behaviors in both susceptible and resilient mice. Thus, in this project, we label them as anxiety/depression (A/D) and anxiety (A) subgroups. An increasing number of studies have implicated the role of mesocorticolimbic ventral tegmental area (VTA) dopamine (DA) reward circuitry in anxiety and depression. Utilizing neural circuit-probing techniques, we previously observed that maladaptive firing activity occurred in the VTA DA neurons projecting to the medial prefront cortex (VTA-mPFC) and VTA DA neurons projecting to the nucleus accumbens (VTA-NAc) selectively in A/D mice (depression-susceptible mice), but not in the A-mice (the depression-resilient group). Our optogenetic studies further demonstrated the causal link between the firing maladaptations in these circuits and depression-related behaviors. However, we strikingly found that the firing activity of VTA neurons projecting to the amygdala (VTA-Amg) was dramatically decreased in both A/D- and A-mice. Based on these unexpected preliminary findings, our central hypothesis is that the VTA-Amg circuit may play a crucial role in mediating the anxiety-like behaviors observed in both A/D- and A-mice following RSDS. To test this, we propose two Specific Aims: (a) to investigate the pathological alterations of VTA-Amg DA circuit neurons in A/D- and A-male and female mice by use of ex vivo brain slice preparation and in vivo optrode recordings from intact animals; and (b) to determine the functional role of VTA-Amg DA neurons in mediating RSDS-induced anxiety behaviors by optogenetically manipulating these circuits in male and female mice. By utilizing these cell type- and circuit-specific electrophysiological and optogenetic techniques, we will determine if a causal relationship exists between the neuronal activity of VTA-Amg DA circuit and anxiety-related behaviors.